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PLoS Negl Trop Dis. 2018 Oct 24;12(10):e0006862. doi: 10.1371/journal.pntd.0006862. eCollection 2018 Oct.

Viridot: An automated virus plaque (immunofocus) counter for the measurement of serological neutralizing responses with application to dengue virus.

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Department of Biology, University of Florida, Gainesville, FL, United States.
Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, Berkeley, CA, United States.
Department of Medicine-Infectious Disease, Vaccine Testing Center, University of Vermont Larner College of Medicine, Burlington, VT, United States.
Mathematical Modelling of Infectious Diseases Unit, Institut Pasteur, Paris, France.
Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States.
Center for Immunization Research, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States.
School of Medicine, University of California, San Francisco, San Francisco, CA, United States.
Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD, United States.
Department of Zoology, University of Cambridge, Cambridge, United Kingdom.
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States.
Emerging Pathogens Institute, University of Florida, Gainesville, FL, United States.


The gold-standard method for quantifying neutralizing antibody responses to many viruses, including dengue virus (DENV), is the plaque reduction neutralization test (PRNT, also called the immunofocus reduction neutralization test). The PRNT conducted on 96-well plates is high-throughput and requires a smaller volume of antiserum than on 6- or 24-well plates, but manual plaque counting is challenging and existing automated plaque counters are expensive or difficult to optimize. We have developed Viridot (Viridot package), a program for R with a user interface in shiny, that counts viral plaques of a variety of phenotypes, estimates neutralizing antibody titers, and performs other calculations of use to virologists. The Viridot plaque counter includes an automatic parameter identification mode (misses <10 plaques/well for 87% of diverse DENV strains [n = 1521]) and a mode that allows the user to fine-tune the parameters used for counting plaques. We compared standardized manual and Viridot plaque counting methods applied to the same wells by two analyses and found that Viridot plaque counts were as similar to the same analyst's manual count (Lin's concordance correlation coefficient, ρc = 0.99 [95% confidence interval: 0.99-1.00]) as manual counts between analysts (ρc = 0.99 [95% CI: 0.98-0.99]). The average ratio of neutralizing antibody titers based on manual counted plaques to Viridot counted plaques was 1.05 (95% CI: 0.98-1.14), similar to the average ratio of antibody titers based on manual plaque counts by the two analysts (1.06 [95% CI: 0.84-1.34]). Across diverse DENV and ZIKV strains (n = 14), manual and Viridot plaque counts were mostly consistent (range of ρc = 0.74 to 1.00) and the average ratio of antibody titers based on manual and Viridot counted plaques was close to 1 (0.94 [0.86-1.02]). Thus, Viridot can be used for plaque counting and neutralizing antibody titer estimation of diverse DENV strains and potentially other viruses on 96-well plates as well as for formalization of plaque-counting rules for standardization across experiments and analysts.

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